genomic.science@science.doe.gov
Office of Biological and Environmental Research
U.S. Department of Energy Office of Science
Susan Gregurick
Office of Biological and Environmental Research
U.S. Department of Energy Office of Science
genomic.science@science.doe.gov
Office of Biological and Environmental Research
U.S. Department of Energy Office of Science
Office of Biological and Environmental Research
U.S. Department of Energy Office of Science
"The BioEnergy Science Center is
focused specifically on the challenge
of overcoming biomass
recalcitrance. If we can manage to
understand and solve this challenge,
we will not only improve our ability
to generate cellulosic ethanol, but
we also will open doors to a vast
array of renewable and sustainable
product pipelines that can serve
many different missions. To address
these important issues, we have
assembled a team of world leaders
in their fields from 20 different institutions and have built a
culture of integration and collaboration that has significantly
accelerated our rate of scientific discovery and application."
– Paul Gilna
Paul Gilna, BESC director, also is deputy director of the Biosciences Division at Oak Ridge National Laboratory. An expert in computational biology, Gilna previously served as executive director of CAMERA (Community Cyberinfrastructure for Advanced Marine Microbial Ecology Research and Analysis), a project providing researchers with bioinformatic tools and services.
Project Description:
The DOE BioEnergy Science Center (BESC), led by Oak Ridge National Laboratory (ORNL) in Oak Ridge, Tennessee, focuses on the fundamental understanding and elimination of biomass recalcitrance— the resistance of cellulosic biomass to enzymatic breakdown into sugars. BESC approaches the problem of biomass recalcitrance from two directions by closely linking (1) plant research to make cell walls easier to deconstruct and (2) microbial research to develop multitalented biocatalysts tailor-made to produce biofuels from this modified plant material in a single step.
Scientists at national laboratories, universities, and private companies that make up the BESC team have extensive experience with studying biomass recalcitrance, and they have made fundamental advances in a wide range of related sciences. The Joint Institute for Biological Sciences systems biology research facility at ORNL serves as the central hub for coordinating research among all BESC partners. BESC's research is organized into three focus areas: (1) Biomass Formation and Modification, (2) Biomass Deconstruction and Conversion, and (3) Characterization and Modeling.
Some recent highlights of BESC research are featured below. By understanding the myriad factors that collectively determine biomass recalcitrance, BESC researchers are providing foundational knowledge that will streamline processing and reduce costs for many different approaches to plant feedstock and cellulosic biofuel production.
Research Strategy
1. Biomass Formation and Modification
BESC biomass formation and modification research
involves understanding the genetics and biochemistry of
plant cell-wall biosynthesis and working directly with two
potential bioenergy crops—switchgrass and poplar—to
develop varieties that are easier to break down into fermentable
sugars. Computational models are being developed to
help BESC researchers identify target genes and successful
strategies for modifying biosynthetic pathways to generate
cell walls that can be readily deconstructed into sugars
for biofuel production. Target genes are turned on or off in
thousands of poplar and switchgrass samples generated and
studied by BESC, and then these samples are characterized to assess how these modifications affect plant cell walls.
These modified plants are growing in greenhouses and at
several sites to help differentiate genetic versus environmental
influences on plant trait development (see figure, Bioenergy
Crop Research at BESC, this page). By understanding
the synthesis and assembly of the polysaccharides and lignin
in plant biomass, BESC researchers are reducing cell-wall
recalcitrance using methods that can be applied to a wide
range of woody and herbaceous plants.
2. Biomass Deconstruction and Conversion
Two key hypotheses drive biomass deconstruction and
conversion research at BESC: (1) microorganisms can be
engineered to enable consolidated bioprocessing (CBP)—
a game-changing, one-step, microbe-mediated strategy
for directly converting plant biomass into ethanol and
(2) enzymes and microbial biocatalysts can be understood
and engineered to synergize with recalcitrance-reducing plant
modifications to achieve better biomass deconstruction.
Model organisms for CBP development include species of Clostridia bacteria that rapidly degrade pure cellulose and then ferment the resulting sugars into ethanol (see figure, BESC Research on Biomass-Deconstructing Microbes). These microbes deconstruct biomass using cellulosomes— multifunctional enzyme complexes that specialize in degrading the mix of complex carbohydrates in cell walls. BESC is studying the structures and activities of these poorly understood multienzyme complexes to design new variants that are more efficient at deconstructing cell walls.
BESC researchers also are investigating microbes that can rapidly degrade biomass at near-boiling temperatures, such as species of Caldicellulosiruptor isolated from hot springs at Yellowstone National Park. By developing genetic tools for these difficult-to-manipulate microbes, BESC is working to engineer novel microbes that add biofuel production capabilities to their native cellulose-degrading abilities. These microbes and their enzymes could provide new biomass-degrading capabilities resistant to the heat and stresses of industrial processing.
Bioenergy Crop Research at BESC. Transformed Populus shoots grow in a greenhouse. These plants are altered in targeted cell-wall pathway genes. [Image courtesy of Oak Ridge National Laboratory]
BESC Research on Biomass-Deconstructing Microbes. Isolated from decaying grass compost, Clostridium cellulolyticum degrades cellulosic biomass using multienzyme complexes called cellulosomes. This scanning electron micrograph shows C. cellulolyticum cells growing on switchgrass biomass. [Photo by Thomas Hass and Shi-You Ding, National Renewable Energy Laboratory]
3. Characterization and Modeling
Advancing BESC goals to develop improved plant materials
and CBP methods that facilitate cost-effective conversion of
biomass to fermentable sugars will require detailed knowledge
of (1) the chemical and physical properties of biomass
that influence recalcitrance, (2) how these properties can
be altered by engineering plant biosynthetic pathways, and
(3) how biomass properties change during pretreatment and
how such changes affect biomass-biocatalyst interactions during
deconstruction by enzymes and microorganisms.
To examine chemical and structural changes that occur in the modified plant cell walls of switchgrass and poplar, BESC analyzes thousands of native and modified biomass samples in a high-throughput screening pipeline that can perform compositional analysis, pretreatment, and enzyme digestibility studies. This screen already has revealed that a few native poplar samples can release most of their sugars using just a mild (hot water) pretreatment. Promising candidates selected from this screening pipeline are passed along to other partner institutions for a variety of detailed chemical, physical, and imaging analyses. The resulting data are incorporated into computational models and simulations used to predict relationships between biomass structure and recalcitrance.
Modeling and simulation tools are part of a knowledgebase BESC is establishing to maintain and share data, materials, experimental processes, and scientific insights across the distributed BESC community. One component of this knowledgebase is a comprehensive set of tools for discovering biomass recalcitrance genes in plant genomes and building pathways for cell-wall synthesis. By extracting and combining results from isolated experiments, this knowledgebase serves as a biological discovery platform for integrating diverse experimental, theoretical, and computational approaches that will help define the genomic and physical bases of plant cell-wall recalcitrance. A public version of the knowledgebase is at http://besckb.ornl.gov.
Translation of BESC Science into
Commercial Applications
Translating BESC research results into the testing of applications
and potential commercial deployment is an important
step toward reaching DOE's bioenergy objectives. BESC has
formed a "commercialization council" of technology-transfer
and intellectual property (IP) management professionals from
partner institutions to evaluate the commercial potential of
new inventions arising from BESC research and to promote
and facilitate the licensing of BESC IP. Inventions are posted
on the center's website (http://bioenergycenter.org/).
Some early inventions address techniques for plant and microbial
genetic transformation, special microscopy methods, and
innovations in biomass sample handling. To build external
relationships that can promote commercialization of new
technologies, BESC provides opportunities for companies
to become BESC Industry Affiliates. Several BESC research
partners and affiliates have pilot facilities for testing BESC
improvements. The University of Tennessee–Genera Energy
and Dupont Danisco Cellulosic Ethanol have established a
demonstration plant for turning corn cobs and switchgrass
into ethanol in Vonore, Tennessee. Mascoma, Verenium,
Ceres, and ArborGen also have pilot and field sites.
Education and Outreach
To prepare the next generation of bioenergy scientists, BESC
provides interdisciplinary research opportunities to graduate
students, postdocs, and visiting scientists. In addition to
these activities in higher education, BESC is teaming with the
Creative Discovery Museum in Chattanooga, Tennessee, to
raise awareness of cellulosic biofuels, carbon emissions from
energy use, and obstacles to a successful biofuel economy.
Targeting fifth-graders, BESC education and outreach efforts
make information accessible to the general public and reach
students when they still are excited about science. Lessons
piloted to thousands of students at schools in Georgia and
Tennessee are available to schools nationwide. BESC also
began "Science Night" programs that build on these classroom
lessons and are offered to students and their families.
The National Geographic Jason Project featured BESC science
in a program developed for high school students. The
BESC website features announcements about BESC outreach
and educational programs, seminars and presentations
describing BESC research, and other resources.
BESC Partners
Location of Center: ORNL Campus, Oak Ridge, Tennessee
Key Targets from a Complex Family of Lignin
Biosynthesis Genes Identified in Switchgrass
Although lignin content and composition have been manipulated
in several plant species by targeting the monolignol
biosynthesis pathway, little is known about the genes and
enzymes associated with this pathway in switchgrass. Cinnamoyl
CoA reductase (CCR) catalyzes the first step in
this pathway dedicated to monolignol synthesis. However,
switchgrass contains numerous copies of CCR-like genes,
complicating the selection of the best gene targets for altering
lignin to reduce cell-wall recalcitrance. By analyzing the
RNA of expressed CCR genes, BESC researchers show that
one of the expressed genes (PvCCR1) encodes an enzyme
actively involved in lignification and thus is a prime target
for down-regulation to improve the degradability and sugar
yield from switchgrass. Ongoing research is investigating how
reducing the expression of the PvCCR1 gene impacts lignin
composition and plant structure. This research was reported
in Escamilla-Treviño, L. L., et al. 2009. "Switchgrass (Panicum
virgatum) Possesses a Divergent Family of Cinnamoyl CoA
Reductases with Distinct Biochemical Properties," New Phytologist
185, 143–55.
Amount of Cellulose Synthase Proteins in Different Subcellular Fractions. The pellet fraction of extracted xylem proteins is enriched in membrane proteins. Significantly higher amounts of a key membrane protein, cellulose synthase, are associated with the pellet fraction relative to the other fractions. [Image from Kalluri et al. 2009]
Thousands of Proteins from Developing Xylem
Cells in Poplar Are Identified
Woody biomass in trees primarily consists of the secondary
cell walls of dead xylem tissue, so developing xylem cells are
useful models for investigating secondary cell-wall formation.
To provide subcellular context for identified protein
functions and to enhance the detection of low-abundance
proteins, subcellular fractionation techniques were used to
obtain crude (soluble protein), pellet (insoluble protein),
and nuclear protein fractions for analysis. Applying an
automated approach known as MudPIT (Multidimensional
Protein Identification Technology), BESC researchers
successfully isolated and identified 6,000 different proteins
from developing xylem cells in the stems of poplar plants.
Results from this project greatly expanded the number of
proteins that had been identified in previous poplar proteome
studies. The protein products of several cell-wall synthesis
genes (e.g., cellulose synthase, sucrose synthase, and
polygalacturonase) were found to be associated with cellular
membranes (see figure), and numerous new candidate genes
for cell-wall synthesis were discovered—many are promising
targets for further functional genomic analysis. Measuring
differences in the whole proteomes of different poplar
variations will increase understanding of the fundamental
properties that underlie the recalcitrance of woody biomass
to degradation. This research was reported in Kalluri, U. C.,
et al. 2009. "Shotgun Proteome Profile of Populus Developing
Xylem," Proteomics 9, 4871–80.
Caldicellulosiruptor bescii on Birchwood Xylan. [Image courtesy of Mike Adams, University of Georgia]
Heat-Tolerant Bacteria Efficiently Degrade
Non-Pretreated Biomass
Presenting the possibility of eliminating the pretreatment step
from cellulosic biofuel production, a hot springs bacterium
known as Caldicellulosiruptor bescii has shown that it can efficiently
degrade crystalline cellulose, xylan (a hemicellulose),
and various types of non-pretreated biomass including hardwoods
such as poplar, high-lignin grasses such as switchgrass,
and low-lignin grasses such as Bermuda grass (see figure). With an optimal growth temperature of 75°C, C. bescii
was able to break down 65% of switchgrass biomass without
pretreatment. This bacterium is the most heat-tolerant biomass
degrader known (withstanding temperatures up to 90°C), and
it primarily produces hydrogen as an end product when grown
on plant biomass. BESC researchers have discovered another
hot springs bacterium (Caldicellulosiruptor obsidiansis), isolated
from Yellowstone National Park, that thrives at 78°C and can
ferment all the simple sugars in cell-wall polysaccharides into
diverse products including ethanol. Combining the functional
capabilities of C. bescii and C. obsidiansis theoretically
could yield organisms that both deconstruct and ferment plant
biomass at temperatures above the boiling point of ethanol
(78.4°C). Producing ethanol in the vapor phase could greatly
reduce the inhibitory effects of ethanol on cell growth. C. bescii
(formerly called Anaerocellum thermophilum DSM 6725) findings
are from Yang, S. J., et al. 2009. "Efficient Degradation of
Lignocellulosic Plant Biomass, Without Pretreatment, by the
Thermophilic Anaerobe 'Anaerocellum thermophilum' DSM 6725," Applied and Environmental Microbiology 75(14), 4762–
69. The discovery of C. obsidiansis was reported in Hamilton-
Brehm, S. D., et al. 2010. "Caldicellulosiruptor obsidiansis sp. nov.,
an Anaerobic, Extremely Thermophilic, Cellulolytic
Bacterium
Isolated from Obsidian Pool, Yellowstone National Park,"
Applied and Environmental Microbiology 76(4), 1014–20.
Validation of Gene Expression in Vascular Tissues. To confirm the expression of identified genes in vascular tissues, probes that specifically bind the mRNA of a small number of the identified genes were applied to switchgrass tissue. The darker regions indicate the vascular bundles where the probes bound the abundant mRNA from the active expression of targeted genes in these regions. [Image courtesy of Elison Blancaflor, The Samuel Roberts Noble Foundation]
Researchers Target Expressed Genes in Vascular
Tissues of Switchgrass
Using a laser-based technique for microdissecting plant tissues,
BESC researchers have targeted and analyzed DNA that is
actively expressed in switchgrass vascular tissues where secondary
cell walls are synthesized and reinforced with lignin. A total
of 2,766 unique genes were identified from 5,734 expressed
DNA segments (known as expressed sequence tags or ESTs). A
significant number of these expressed sequences are novel with
no significant hits to existing EST data. A small subset of the
identified genes was targeted with labeled probes to visualize the
expression of these genes in live plant tissue (see figure),
and researchers found that several genes have much higher
expression in the vascular bundles. The gene list generated from
this study provides an important genomic resource for narrowing
the range of molecular targets that could play key roles in
modifying the lignin content of switchgrass and other related
bioenergy crops. This research was reported in Srivastava, A. C.,
et al. 2010. "Collection and Analysis of Expressed Sequence Tags
Derived from Laser Capture Microdissected Switchgrass (Panicum
virgatum L. Alamo) Vascular Tissues," BioEnergy Research,
DOI: 10.1007/s12155-010-9080-8.
New Strategy Enhances Microbial Resistance
to Inhibitory Pretreatment Chemicals
The chemical and physical processes for pretreating biomass
help unravel the complex matrix of cell-wall components
and enhance enzyme accessibility to these materials, but
pretreatments also generate chemicals such as acetate that
inhibit sugar fermentation to biofuels. Using a combination
of adaptation, genetic engineering, and systems biology tools,
BESC researchers have developed acetate-resistant strains of
two industrial ethanol producers (the bacterium Zymomonas
mobilis and the yeast Saccharomyces cerevisiae) by changing the
expression of genes encoding transport proteins that move
substances across the cell membrane. These proteins (called
antiporters) transport proton and sodium ions and form gradients
that are adversely impacted by the presence of acetate.
By resequencing a Z. mobilis strain
that had been adapted to withstand
high acetate concentrations, BESC
researchers discovered specific
mutations in antiporter genes
that enable acetate resistance. The
specific antiporter mutations were
validated using genetically engineered
Z. mobilis and yeast showing
the broad impact of these changes.
This research is reported in Yang, S.,
et al. 2010. "Paradigm for Industrial
Strain Improvement Identifies
Sodium Acetate Tolerance Loci in
Zymomonas mobilis and Saccharomyces
cerevisiae," Proceedings of
the National Academy of Sciences
107(23), 10395-400.
